BIOCHEMICAL CHARACTERIZATION OF SYNAPTOSOMAL FUNCTION AND NEURAL REGENERATION IN THE RODENT OLFACTORY PATHWAY.
Item
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Title
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BIOCHEMICAL CHARACTERIZATION OF SYNAPTOSOMAL FUNCTION AND NEURAL REGENERATION IN THE RODENT OLFACTORY PATHWAY.
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Identifier
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AAI8203317
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identifier
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8203317
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Creator
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ROCHEL, SARAH.
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Contributor
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Frank L. MargoliS
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Date
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1981
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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The purpose of this project is to study and elucidate biochemical mechanisms of the chemoreceptor neurons in the olfactory pathway.;The olfactory chemoreceptor neuron reconstitution was studied via the activity of ornithine decarboxylase (ODC) as a monitor of cellular regeneration. ODC activity in olfactory tissue (0.2-0.4 nmol/mg pr/hr) was 10 to 30 times higher than in other cerebral tissues. ODC activity in the olfactory mucosa declined within 3 hours after degeneration of chemoreceptor neurons was induced surgically or chemically. ODC activity recovered when chemoreceptor differentiation from stem cells occurred. This implies that ODC activity is, in part, located in chemoreceptor neurons. Furthermore, change in ODC activity was the first event detected in response to degeneration and regeneration inducing treatments. As such, it may prove useful as a reconstitution marker of chemoreceptor neurons.;A second goal of this study was to provide biochemical evidence that the dipeptide carnosine ((beta)-alanyl L-histidine) acts as a neurotransmitter. Specifically, its release from olfactory nerve endings in response to depolarizing stimuli, has been established providing an essential requirement for its classification as a neurotransmitter.;Synaptosomes were prepared from olfactory bulbs of mice. DNA and nuclei were excluded from these fractions, which contained mostly multi (P(,1)) and mono (P(,2)) synaptosomal structures. These fractions contained carnosine, enzymes synthesizing the neurotransmitters GABA (GAD) and dopamine (TH), and binding sites for bulbar neurotransmitters.;Membrane potentials ((DELTA)(PSI)) of the synaptosomes were characterized using the lipophilic cation tetraphenylphosphonium (TPP('+)) as a probe, and the conditions for (DELTA)(PSI) measurements in synaptosomes were established. TPP('+) was found to accumulate in the intrasynaptosomal mitochondria, causing dissipation of their (DELTA)(PSI) in proportion to its concentration. The mitochondrial accumulation of TPP('+) was eliminated using oligomycin/argon. Under these conditions TPP('+) accumulation provides direct evaluation of the plasma membrane (DELTA)(PSI). (DELTA)(PSI) of olfactory bulb synaptosomes was consequently calculated to be -65 and -77 mV in P(,1) and P(,2) fractions, respectively. (DELTA)(PSI) was dependent on potassium concentration gradient across the membrane. Synaptosomal (DELTA)(PSI) was maintained by the Na, K, ATPase-pump, indicated by the ouabain induced extensive depolarization accompanied by potassium loss, one hour after its application. The synaptosomes maintained an action potential sodium gate, as indicated by veratridine induced depolarization, and its blockade by tetrodotoxin.;The release of in vivo synthesized carnosine from synaptosomes was studied in an in vitro superfusion system. Carnosine is localized solely in the nerve endings subfraction which derive from chemoreceptors. Therefore, its efflux corresponds to the specific properties of these cells. Carnosine was observed to be relatively immobilized in the synaptosomes, similar to catecholamines and GABA, and unlike nontransmitter amino acids which are characterized by high efflux rates. Carnosine was released from synaptosomes through two mechanisms. The first, calcium independent spontaneous efflux detected under nondepolarizing conditions. The second, a calcium dependent, depolarization-stimulated release. This latter process was stimulated by 60 mM K('+), or by veratridine. The sodium gate inhibitor tetrodotoxin reversed the veratridine effect. Cytoplasmic carnosine exchange with external carnosine did not affect the 60 mM K('+) induced release, hence it must represent efflux by a mechanism different than the depolarization induced one.;The results of this study are consistent with the model suggesting the packaging of carnosine in synaptic vesicles, and its release by exocytosis upon exposure to depolarizing stimuli.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
